Project Summary/Abstract The spatial and temporal coordination of multiple proteins is critical for the regulation of complex processes in bacterial cells, including peptidoglycan synthesis for cell elongation and cell division, morphogenesis, cell differentiation, biogenesis of external structures, adhesion to surfaces and biofilm formation, and aging. The main goal of this study is to determine the mechanisms that control the spatio-temporal organization of bacterial cells and how this organization is translated into phenotypes that benefit fitness. The project has three major parts: 1. A study the mechanisms that control the spatio-temporal dynamics of peptidoglycan synthesis in different zones to drive cell elongation, division, and morphogenesis. Fluorescent probes that label the sites of peptidoglycan synthesis will be optimized to enable experiments with increased spatial and temporal resolution. Septal peptidoglycan synthesis patterns will be studied by successive labeling with peptidoglycan probes of different colors, whose spatial pattern will provide a chronological account of the areas of PG synthesis during each pulse labeling. The effect of varying FtsZ threadmilling and the movement of the PBP2b septal PG synthesis protein on the velocity of peptidoglycan synthesis will be tested. The function of the SpmX morphogen, which specifies small zones of peptidoglycan synthesis to generate thin cylindrical extensions of the cell envelope called stalks, will be studied by determining its structure, its localization mechanisms, and by identifying interacting proteins. 2. A study of the mechanisms by which protein localization and cellular asymmetry regulate the cell cycle, cell differentiation, and aging. A novel mechanism of regulation of histidine kinases by dephosphorylation by the polar scaffold protein PodJ will be investigated using biochemical approaches and mutagenesis to determine its the mechanism. The mechanism of PodJ localization to the pole and its degradation to release inhibition of the histidine kinase will be studied. The role of cellular asymmetry in aging will be determined by studying its impact on damage segregation. 3. A study of the mechanisms of bacterial adhesion to surfaces and the biochemical properties of a strong adhesive. The role of the Caulobacter crescentus flagellum and pili in surface sensing and in mediating the transition from the reversible to the permanent phase of adhesion, culminating in the synthesis of an adhesive holdfast, will be studied by their quantitative tracking during the adhesion process of various mutants. The biophysical basis for the impressive strength of the holdfast adhesive will be studied by atomic force microscopy dynamic force spectroscopy and high resolution analysis of its structure by a combination of E-beam etching or ion beam milling, AFM imaging, and nanoindentation. Insights gained from these studies can be used to design strategies to inhibit growth, prevent key morphological changes, or alter important protein localization pathways in pathogens, thereby improving our ability to control them.